I have recently identified a novel vinculin-binding protein, paxillin. This proposal is aimed at further characterizing paxillin, its interaction with vinculin and at identifying other paxillin-binding proteins. These data are expected to contribute to our understanding of cytoskeletal- membrane interactions. The nucleotide sequence of paxillin will be obtained from paxillin cDNA. The resulting predicted amino acid sequence will be analyzed for potential homologies with other proteins and for suggestions of possible enzymatic or structural function. Appropriate biochemical assays will be performed on the purified protein to confirm/disprove such indications. The paxillin cDNA will be transfected in whole or in part into mammalian cells to determine which regions of the protein are required for focal adhesion localization. In parallel, partial or full length fusion proteins of paxillin and vinculin will be used in in vitro assays to map their binding sites. The interaction of these two proteins will also be examined at the electron microscope level. Following transformation by Rous sarcoma virus, paxillin contains high levels of phosphotyrosine. The role of paxillin phosphorylation in the loss of focal adhesion integrity during viral or chemically induced transformation will be investigated. The effects of paxillin phosphorylation on vinculin binding will be determined in vitro. In an attempt to identify novel paxillin-binding proteins I will isolate protein complexes containing paxillin from cells grown in culture possessing focal adhesions at their interface with the substratum. Cells will be gently permeabilized and, in some cases, the actin cytoskeleton stabilized with phalloidin. Proteins will be differentially extracted from these cells using changes in ionic strength, pH and divalent cation concentrations. Proteins released in conjunction with paxillin will be co-isolated by immunoprecipitation of paxillin under non-denaturing conditions. Where appropriate, experiments will be performed in the presence of chemical cross-linkers to stabilize interactions between paxillin and its associated proteins. Antibodies will be generated against any novel paxillin-binding proteins and these will be used to study further the proteins regarding their role in cytoskeletal membrane organization.